Summary

Clone O40 Encodes a Cytochrome P450 Mixed Function Monooxygenase

The largest ORF of clone O40 encodes a cytochrome P450 monooxygenase of predicted molecular weight 48 kD and a pI of 6.62. The cytochrome P450 monooxygenases constitute a large gene superfamily of membrane-bound enzymes which catalyze the oxidation of diverse and often overlapping substrates of both endogenous and xenobiotic origin in diverse organisms from bacteria to fungi, plants and animals. O40 is the first orchid cytochrome P450 identified, and by convention, it represents the second member of the CYP78 gene family and has consequently been named CYP78A2 by the Cytochrome P450 Gene Nomenclature Committee. For details of the degree of amino acid sequence identity of the O40 protein CYP78A2 to other plant cytochrome P450 monooxygenasesis please see our publication (Nadeau, Zhang, Li and O'Neill, 1996).

Analysis of Gene Expression

In situ hybridization of clone O40 to transverse sections of the orchid ovary revealed that, contrary to our initial expectations based on RNA blot hybridization results, clone O40 is expressed exclusively in the pollen tubes. The identification of this gene by differential screening for ovule-specific cDNAs is not completely unexpected, as pollen tubes become intertwined with the ovules in the ovary and consequently contribute to the RNA population harvested despite our attempts to remove them. Panels A and B illustrate tissue containing ovules at the archesporial stage of development, including pollen tubes which are growing in the locule adjacent to the specialized placental epidermis. No pollen tubes are present on the other side of the placenta, which does not differentiate these specialized epidermal cells. These panels clearly illustrate the very high level of O40 transcript present in the pollen tubes, as compared to Panels C and D which show sections from the same tissue block probed with the sense-strand RNA as a control. The cytoplasm of the majority of pollen tubes in the area is localized near the placental surface while the bulk of tissue further out in the locule is composed of empty pollen tubes left behind as the tips elongate.